Effects of arsenobetaine a major organic arsenic compound in seafood on the maturation
and functions of human peripheral blood monocytes macrophages and dendritic cells.

код для вставки на сайт или в блог

ссылки на документ

APPLIED ORGANOMETALLIC CHEMISTRY
Appl. Organometal. Chem. 2004; 18: 431–437
Bioorganometallic
Published online in Wiley InterScience (www.interscience.wiley.com). DOI:10.1002/aoc.690
Chemistry
Effects of arsenobetaine, a major organic arsenic
compound in seafood, on the maturation and functions
of human peripheral blood monocytes, macrophages
and dendritic cells
Takami Ohta, Teruaki Sakurai* and Kitao Fujiwara
Laboratory of Environmental Chemistry, School of Life Science, Tokyo University of Pharmacy and Life Science, Horinouchi 1432-1,
Hachioji, Tokyo 192-0392, Japan
Received 14 October 2003; Revised 15 December 2003; Accepted 10 February 2004
We examine the in vitro immunotoxicity of synthetically pure arsenobetaine [AsBe; trimethyl
(carboxymethyl) arsonium zwitterion], which is a major organic arsenic compound in seafood, on
various human immune cells, such as peripheral blood monocytes, monocyte-derived macrophages
and monocyte-derived dendritic cells (DCs). In particular, we examine the differentiation of monocytes
into macrophages or DCs by comparing the effects of AsBe with those pentavalent inorganic arsenate.
AsBe neither enhanced nor inhibited the differentiation of human monocytes into macrophages
or DCs, and also did not affect their various immune functions. Furthermore, AsBe had no
cytolethality in monocyte-derived macrophages or DCs even at a concentration of 5 mmol l−1 . In
contrast, inorganic arsenate showed strong cytolethality in these human immune cells in vitro at
micromolar concentrations. These data indicate that the organic arsenic compound AsBe in seafood
has no in vitro immunotoxicity in human immune cells. Copyright  2004 John Wiley & Sons, Ltd.
KEYWORDS: arsenobetaine; arsenic; seafood; human; monocyte; differentiation; macrophage; dendritic cell; immunotoxicity;
marine animal
INTRODUCTION
Arsenic has been considered to be a poison for a long time.
Since the 1820s, it has been generally accepted as a potent
environmental carcinogen for some human malignancies.1 It
has previously been reported that common marine animals,
which are ingested daily as seafood in many countries, contain
very high concentrations of arsenicals, ranging from about
4 to 80 µg g−1 ,2 and that these arsenicals are commonly in
the form of water-soluble organic arsenic compounds. The
limit for arsenic in drinking water in Japan is 10 µg l−1 . If
this limit was applied to seafood, as 10 ng g−1 , most of
them would be deemed unfit for consumption, given that
their contents are often 1000 times this concentration.3 This
finding has caused great concern with respect to the health of
people who often ingest considerable amounts of seafood. In
*Correspondence to: Teruaki Sakurai, Laboratory of Environmental
Chemistry, School of Life Science, Tokyo University of Pharmacy and
Life Science, Horinouchi 1432-1, Hachioji, Tokyo 192-0392, Japan.
E-mail: sakurai@ls.toyaku.ac.jp
particular, it is very important for us to clarify the biological
effects of the trimethyl (carboxymethyl) arsonium zwitterion,
namely arsenobetaine (AsBe), which is a major organic arsenic
compound in marine animals.4 However, there have only
been a few reports about them because sufficient amounts of
pure AsBe for biological experiments have not been obtained.
In 1985, investigation of the acute toxicity of AsBe
using synthetic pure material clarified that it had no acute
toxicity in murine models even over 10 g kg−1 when it
was administered orally.5 Subsequently, we showed that
AsBe had no immunotoxicity in vitro in murine immune
effector cells, such as mouse peritoneal macrophages, alveolar
macrophages and splenocytes,6 and we also demonstrated
that AsBe was not toxic in vitro to various mammalian
cell lines, such as mouse macrophage RAW264.7 cells, rat
liver TRL1215 cells and human skin TIG-112 cells, even
over 20 mmol l−1 .7 Furthermore, we recently reported that
AsBe showed a unique biological effect on murine bone
marrow (BM) cells in vitro; AsBe significantly enhanced the
initial adhesion ability and viability of immature mouse BM
Copyright  2004 John Wiley & Sons, Ltd.
432
T. Ohta T. Sakurai and K. Fujiwara
cells and subsequently increased the continuous survival
of Mac-1 (CD11b) positive large spreading cells, especially
granulocytes and macrophages, that originated from the BM
cells.8 These data imply that AsBe has a possible application to
enhance some functions of immature immune cells. However,
the reasons why AsBe enhances the survival of immature BM
cells are not yet precisely clarified, and there are no data about
the effects of AsBe on human immune cells.
Human peripheral blood monocytes are one of the immature immune effector cells and precursors of mature immune
effector cells in the immune system. Monocytes can differentiate into macrophages by the granulocyte/macrophage
colony stimulating factor (GM-CSF) or macrophage colony
stimulating factor (M-CSF), and into dendritic cells (DCs)
by the combination of GM-CSF plus interleukin-4 (IL-4).9
Macrophages are one of the principal immune effector cells
that play essential roles as secretory, phagocytic and antigenpresenting cells.10 DCs are the major antigen-presenting cells
capable of strongly stimulating primary T-cell responses.9,11
In this study, we observed the effects of AsBe on human
monocytes, monocyte-derived macrophages and monocytederived DCs, mainly concerning the differentiation of human
monocytes into macrophages and/or DCs in vitro.
EXPERIMENTAL
Arsenic
AsBe was synthesized from trimethylarsine by reaction with
ethyl β-bromo-propionate in an atmosphere of CO2 (Tri
Chemical Laboratory Inc., Yamanashi, Japan), and was twice
recrystallized from acetone containing a trace of methanol.5
It produced white prismatic crystals, with a melting point at
204 ◦ C. Its structure was confirmed by 1 H nuclear magnetic
resonance (NMR), 13 C NMR, high-performance liquid chromatography–inductively coupled plasma mass spectrometry
(HPLC–ICP-MS) and fast-atom bombardment MS. The purity
of this synthesized AsBe was >99.9% as determined by thinlayer chromatography, HPLC–ICP-MS and gas chromatography–MS.5 – 8 The synthesized AsBe was treated with Kurimover I (Kurita Water Industries Ltd, Tokyo, Japan), which
is a removal agent for lipopolysaccharide (LPS), and LPS
contamination of this AsBe was <0.000 04%(wt wt−1 ) as determined by the endotoxin-specific limulus test. Sodium arsenate
was purchased from Wako Pure Chemical Co. (Osaka, Japan),
and was twice recrystallized from methanol. LPS contamination of this arsenate was <0.000 000 2%(wt wt−1 ).
Bioorganometallic Chemistry
monocytes were suspended in RPMI 1640 medium (Sigma
Chemical Co., St Louis, MO, USA) containing 10% heatinactivated fetal calf serum, 100 U l−1 penicillin, and 100 µg l−1
streptomycin (FCS-RPMI). Monocytes plated in flat-bottomed
96-well tissue culture plates (2 × 104 or 2 × 105 per well) or 12well tissue culture plates (5 × 105 per well) were then cultured
with 5000 U ml−1 recombinant human (rh) GM-CSF (PeproTech EC Ltd, London, UK), 1000 U ml−1 rhM-CSF (Genzyme
Tech Co., Boston, MA, USA) or rhGM-CSF plus 2000 U ml−1
rhIL-4 (Genzyme) in the presence or absence of arsenic.
Cultures were maintained in a humidified atomosphere of
5%CO2 –95% air at 37 ◦ C. Cells which were incubated with
rhGM-CSF or rhM-CSF for 6 days were differentiated into
GM-type macrophages (GM-Mps) or M-type macrophages
(M-Mps) respectively.9,10 Cells which were incubated with
rhGM-CSF plus rhIL-4 for 6 days were differentiated into
DCs.9,10
Assay for cellular viability
The cellular viability was determined by measuring live
cells using the AlamarBlue (AB) assay, which is similar to
the methylthiazoletetrazolium assay. Briefly, cells (2 × 104
or 2 × 105 /100 µl per well) were incubated on flat-bottomed
96-well tissue culture plates several times. At 3 h before
the end of the incubation, 10 µl per well of AB solution
(Iwaki Glass Co., Chiba, Japan) was added directly to the
wells, and the absorbance at 570 nm (reference 600 nm) was
measured using a microplate reader model 550 (Bio-Rad
Laboratories, Hercules, CA, USA). Arsenic itself did not affect
the absorbance of the AB solution, even at concentrations over
40 mmol l−1 .
Determination of the number and species of
cells
Monocytes (2 × 104 or 2 × 105 per well) were incubated with
various concentrations and combinations of rhGM-CSF,
rhM-CSF, rhIL-4 or arsenic on flat-bottomed 96-well tissue
culture plates for 6 days at 37 ◦ C using FCS-RPMI. After
the incubation, the number and the species of cells in
the wells were determined under a microscope equipped
with an eyepiece micrometer at ×200 magnification, whose
microscopic field was equivalent to 0.25 mm2 . M-Mps are
elongated and have a spindle-like morphology, and GM-Mps
are round with a saucer-like morphology.9,10 DCs induced
by rhGM-CSF plus rhIL-4 displayed a dendritic morphology
with delicate membrane projections that were veil-like or
sheet-like processes.9
Phagocytosis assay
Human peripheral bloood monocytes
Human peripheral blood monocytes were obtained from normal healthy volunteers by centrifugation on a Lymphoprep
(Nycomed, Oslo, Norway) gradient. Monocytes were separated with anti-CD14 monoclonal antibody (mAb)-coated
MicroBeads using MACS single-use separation columns
(Miltenyi Biotec, Bergisch Gladbach, Germany). Purified
Copyright  2004 John Wiley & Sons, Ltd.
Functions of monocyte-derived macrophages were assessed
by phagocytic ability using zymosan particles. Cells were
incubated with zymosan (Sigma) in FCS-RPMI at a ratio of
40 particles per cell on flat-bottomed 96-well tissue culture
plates for 30 min at 37 ◦ C. After the incubation, the cells
were stained using Diff-Quick stain kit (Kokusai Shiyaku
Co., Hyogo, Japan), and the number of cells ingesting more
Appl. Organometal. Chem. 2004; 18: 431–437
Bioorganometallic Chemistry
than three particles and phagocytosed particles per cell were
determined by counting at least a total of 300 cells under the
microscope. Phagocytic activity is expressed as the number
of phagocytosed particles per cell which intook the particles.
Mixed leukocyte reactions
DC function was assessed by mixed leukocyte reactions
(MLRs) assay. CD14− CD4+ naive T cells were purified by
negative selection of human blood peripheral monocytes
using anti-CD14 mAb-coated MicroBeads and positive
selection using anti-CD4 mAb-coated MicroBeads (Miltenyi
Biotec). DCs or GM-Mps (stimulators; 5 × 103 per well) were
incubated with allogeneic or autologous naive T cells
(2 × 105 per well) on flat-bottomed 96-well tissue culture
plates in 200 µl per well for 6 days at 37 ◦ C; then, the
proliferative response of T cells was determined by AB
assay. Briefly, 6 h before the end of the incubation, 20 µl
of AB solution was added directly to the 96-well plates, and
the absorbance at 570 nm (reference 600 nm) was measured
using a microplate reader model 550. Data are expressed as
percentage absorbance using the values from T cells incubated
without stimulators as 100%.
Assay for tumor necrosis factor α production
Monocytes (2 × 104 per well) were incubated with 1000 U
ml−1 M-CSF or 5000 U ml−1 GM-CSF on 96-well tissue culture
plates for 6 days at 37 ◦ C. After the incubation, the cells were
exposed to various concentrations of AsBe in the presence
or absence of 1 ng ml−1 LPS (0111; B4; Sigma) for 2 days at
37 ◦ C. The tumor necrosis factor α (TNFα) concentrations
in the culture supernatants were quantitated by a doublesandwich enzyme-linked immunosorbent assay (ELISA). A
96-well plate was coated with mouse anti-human TNFα mAb
(Genzyme) in phosphate-buffered saline (PBS). Uncoupled
binding sites in the wells were blocked with PBS containing
1% bovine serum albumin, 5% sucrose, and 0.05% sodium
azide. The wells were incubated with 50 µl of samples in
triplicate for 40 min at 37 ◦ C and then exposed to biotinylated
goat anti-human TNFα polyclonal Ab (Genzyme). The
plate was developed using a peroxidase-labeled streptavidin
(Nichirei Co., Tokyo, Japan) and peroxidase substrate (TMB
microwell peroxidase substrate system, Kirkegadd & Perry
Lab., Gaithersburg, MD, USA). The absorbance at 450 nm
(reference 630 nm) was measured using a microplate reader
model 550 (Bio-Rad Laboratories). Aliquots of rhTNFα
(Genzyme) were used to construct a standard curve, and the
results were calculated as amounts of TNFα per microgram
of cellular protein determined by BCA protein assay reagent
(Piece Co., Rockford, IL, USA) with bovine serum albumin as
the standard.
Arsenic analysis
Monocytes (1 × 106 per well) were incubated with 5 mmol
ml−1 AsBe or arsenate on flat-bottomed 12-well tissue culture
plates for 24 h at 37 ◦ C. After the incubation, the cells were
rinsed and lysed with 0.3 ml water. Then, 3 ml of nitric acid
Copyright  2004 John Wiley & Sons, Ltd.
Arsenobetaine effects in human cells
and 1 ml of sulfuric acid were added to the cell lysates and
heated at 240 ◦ C until dense fumes of sulfur trioxide appeared.
The digested solutions were neutralized with ammonium
hydroxide, followed by water to bring the solution volume up
to 5 ml. 1 ml of hydrochloric acid, 0.5 ml of 20% ascorbic acid
and 0.5 ml of 20% potassium iodide were then added to the
solutions. The total arsenic amount was analyzed by hydride
generation coupled with atomic absorption spectrometry
using SpestraAA-220 (Varian Australia Pty Ltd., Mulgrave
Victria, Australia). The results are expressed as the cellular
arsenic content per gram cellular proteins determined by BCA
protein assay.2,12,13
Statistics
Statistical evaluations in experiments were expressed as
the arithmetic mean plus/minus the standard error of the
mean (SEM) and performed by analysis of variance followed
by Dunnett’s multiple comparison test or Tukey’s honestly
significant difference test. A value of p < 0.05 was considered
significant in all cases.
RESULTS
Effect of AsBe on the differentiation into
macrophages from human monocytes
M-CSF and GM-CSF are potent cytokines which induce differentiation and proliferation of monocytes into macrophages.
Human monocytes can differentiate into two morphologically
distinct types of macrophage, i.e. M-Mp and GM-Mp, by MCSF and GM-CSF respectively. We examined the effects of a
pentavalent organic arsenic AsBe on these maturations from
monocytes into macrophages, comparing it with the effects
of a pentavalent inorganic arsenic arsenate. Monocytes were
incubated with M-CSF or GM-CSF in the presence or absence
of various concentrations of AsBe or arsenate for 6 days at
37 ◦ C; then, the numbers of monocyte-derived macrophages
were counted. As shown in Fig. 1A and B, AsBe did not affect
the number of monocyte-derived M-Mps and GM-Mps even
at a concentration of 5 mmol l−1 , although arsenate significantly decreased it; its inhibitory concentration in vitro in 50%
of a population (IC50 ) was 280 µmol l−1 or 180 µmol l−1 in
M-Mps or GM-Mps respectively. In morphological investigations, AsBe also did not affect the morphological changes in
either type of monocyte-derived macrophage. Additionally,
we investigated the cellular viability of monocyte-derived
macrophages exposed to arsenic during their differentiations
by the AB assay. As shown in Fig. 1C and D, AsBe had no
effect on their viability, although arsenate showed strong
cytolethality on them; its lethal concentration in vitro in 50%
of a population (LC50 ) was 200 µmol l−1 , and most cells were
killed by millimolar concentrations of arsenate.
Phagocytic ability is one of the principal immune functions
of macrophages.10 Table 1 shows the phagocytic ability of
monocyte-derived macrophages exposed to AsBe during their
differentiations. Monocytes were incubated with M-CSF or
Appl. Organometal. Chem. 2004; 18: 431–437
433
Bioorganometallic Chemistry
T. Ohta T. Sakurai and K. Fujiwara
M-Mps
GM-Mps
350
300
250
200
a
150
100
a
50
a
0
100
0
1000
Number of GM-Mps (cells mm-2)
B
AsBe
arsenate
400
10000
C
AsBe
arsenate
1200
1000
800
600
400
a
200
a
a
0
0
100
1000
10000
D
AsBe
arsenate
120
100
80
60
40
20
a
a
a
0
0
100
1000
10000
Arsenic concentration (µmol dm-3)
Metabolic integrity (% Control)
Number of M-Mps (cells mm-2)
A
Metabolic integrity (% Control)
434
AsBe
arsenate
120
100
80
a
60
40
a
20
a
a
0
0
100
1000
10000
Arsenic concentration (µmol dm-3)
Figure 1. Effect of arsenic on the differentiation into macrophages from monocytes. Monocytes (2 × 104 per well) were incubated
with M-CSF (A, C) or GM-CSF (B, D) in the presence or absence of various concentrations of AsBe ( ), arsenate () or medium alone
on 96-well tissue culture plates for 6 days at 37 ◦ C. (A, B) The number and species of monocyte-derived macrophages (A, M-Mps; B,
GM-Mps) were counted. (C, D) The cellular viability of monocyte-derived macrophages (C, M-Mps; D, GM-Mps) was determined by
AB assay. One representative experiment out of five similar ones is shown. The results are expressed as arithmetic mean plus/minus
SEM of triplicate dishes. a p < 0.001 comparison with monocytes incubated without arsenic.
°
Table 1. Effect of AsBe on the phagocytic activity of
monocyte-derived macrophagesa
Cytokine
M-CSF
GM-CSF
AsBe
(5 mmol l−1 )
Percentage
of phagocytes
Phagocytic
activityb
−
+
−
+
71.2 ± 1.5
74.2 ± 7.6
76.3 ± 5.3
69.4 ± 3.1
4.84 ± 0.33
5.22 ± 0.53
6.37 ± 0.63
7.28 ± 0.23
a
Monocytes were incubated with M-CSF or GM-CSF in the presence
or absence of AsBe (5 mmol l−1 ) for 6 days at 37 ◦ C. After the
incubation, monocyte-derived macrophages were further incubated
with zymosan at a ratio of 40 particles per cell for 30 min at
37 ◦ C. The numbers of cells ingesting more than three particles
and phagocytosed particles were determined by counting at least 300
cells under a microscope. One representative experiment out of three
similar ones is shown. The results are expressed as arithmetic mean
plus/minus SEM of triplicate dishes.
b Phagocytic activity is the number of phagocytosed particles per cell
which intook the particles.
Copyright  2004 John Wiley & Sons, Ltd.
GM-CSF in the presence or absence of 5 mmol l−1 AsBe for
6 days at 37 ◦ C. The phagocytic activity of the cells was then
assessed, and AsBe was found to have no effect on the
phagocytic ability of these macrophages.
Effect of AsBe on the differentiation into DCs
from human monocytes
Using the combination of GM-CSF and IL-4, human
monocytes can differentiate into DCs, which are the major
antigen-presenting cells capable of stimulating primary Tcells responses.9,11 We examined the effects of AsBe on this
maturation from monocytes into DCs by comparison with the
effects of arsenate. Monocytes were incubated with GM-CSF
plus IL-4 in the presence or absence of various concentrations
of AsBe or arsenate for 6 days at 37 ◦ C; then, the number of
monocyte-derived DCs were counted. As shown in Fig. 2A,
AsBe did not affect the number of monocyte-derived DCs
even at a concentration of 5 mmol l−1 , although arsenate
significantly decreased it; its IC50 value was 590 µmol l−1 .
Appl. Organometal. Chem. 2004; 18: 431–437
Bioorganometallic Chemistry
Table 2. Effect of AsBe on MLRs of monocyte-derived DCa
A
Number of DCs (cells mm-2)
Arsenobetaine effects in human cells
AsBe
arsenate
250
200
Stimulator
150
GM-Mps
DCs
100
a
50
b
0
100
1000
B
10000
AsBe
arsenate
120
100
80
AsBe (5 mmol l )
Allogeneic
Autologous
−
−
+
115.6 ± 4.4
193.9 ± 7.9c
219.1 ± 8.4c
68.5 ± 8.5
142.8 ± 16.1d
124.2 ± 1.3e
a
0
Metabolic integrity (% Control)
MLRb (% control)
−1
a
60
Monocytes were incubated with GM-CSF or GM-CSF plus IL-4 in
the presence or absence of AsBe (5 mmol l−1 ) for 6 days at 37 ◦ C.
After the incubation, the cells (stimulators; GM-Mps or DCs) were
further incubated with allogeneic or autologous T cells for 6 days at
37 ◦ C. The proliferative response of the T cells was then determined
by AB assay. One representative experiment out of four similar ones
is shown. The results are expressed as arithmetic mean plus/minus
SEM of triplicate dishes.
b Data are expressed as percentage absorbance using the values from
T cells incubated without stimulators as 100%.
c p < 0.001, comparison with T cells incubated with GM-Mps as a
stimulator.
d p < 0.01.
e p < 0.05.
b
40
20
0
0
100
1000
10000
stimulated the T-cell responses, and the addition of AsBe
during the differentiation of monocytes into DCs did not
affect this immune function of DCs.
Arsenic concentrations (µmol dm-3)
Figure 2. Effect of arsenic on the differentiation into DCs
from monocytes. Monocytes (2 × 104 per well) were incubated
with GM-CSF plus IL-4 in the presence or absence of various
concentrations of AsBe ( ), arsenate () or medium alone
on 96-well tissue culture plates for 6 days at 37 ◦ C. (A) The
numbers of monocyte-derived DCs were counted. (B) The
cellular viability of monocyte-derived DCs was determined by
AB assay. One representative experiment out of five similar
ones is shown. The results are expressed as arithmetic mean
plus/minus SEM of triplicate dishes. a p < 0.05 comparison with
monocytes incubated without arsenic. b p < 0.01.
°
When the cellular viability of monocyte-derived DCs exposed
to AsBe during their differentiations was determined by the
AB assay, AsBe also had no effect on the cellular viability,
although arsenate showed significant cytolethality with a
LC50 value of 1.6 mmol l−1 , and most cells were killed by
millimolar concentrations of arsenate (Fig. 2B).
The most important characteristic of DCs is their strong
T-cell stimulating activity.9 DCs, but not GM-Mps, stimulate
both allogeneic and autologous T cells in MLRs.9,14 Table 2
shows the effect of AsBe on the T-cell stimulating activity
(MLRs) of monocyte-derived DCs exposed to AsBe during
their differentiations. Monocytes were incubated with GMCSF plus IL-4 in the presence or absence of 5 mmol l−1 AsBe
for 6 days at 37 ◦ C; the T-cell stimulating activity of these cells
was then determined by MLRs assay. As a result, monocytederived DCs, but not GM-Mps (negative control), strongly
Copyright  2004 John Wiley & Sons, Ltd.
Effect of AsBe on the viability and functions of
human monocyte-derived macrophages and
DCs
We examined the cytotoxic effects of AsBe using human
mature immune effector cells, such as human monocytederived macrophages and DCs, by comparison with the
effects of arsenate. Monocyte-derived M-Mps, GM-Mps or
DCs were incubated with various concentrations of AsBe or
arsenate for 2 days at 37 ◦ C; the cellular viability was then
determined by the AB assay. As shown in Fig. 3, AsBe had
no cytolethality on these mature immune effector cells, even
at a concentration of 5 mmol l−1 , although arsenate showed
significant cytolethality in these cells with LC50 values of
850 µmol l−1 –1.9 mmol l−1 , and most cells were killed by
millimolar concentrations of arsenate.
Additionally, we investigated the effects of AsBe on the
functions of mature immune effector cells. Macrophages are
known to be very sensitive to changes in environmental
conditions and release various immune inflammatory factors
when they are stimulated.12,15,16 Figure 4 shows the effect
of AsBe on the TNFα secretion, which is one of the
potent inflammatory cytokines,12,15 in monocyte-derived
macrophages. M-Mps and GM-Mps were incubated with
various doses of AsBe in the presence or absence of 1 ng ml−1
LPS for 2 days at 37 ◦ C; the concentrations of TNFα in the
culture supernatants were measured by double-sandwich
ELISA. As a result, AsBe had no effect on the release of TNFα
when the macrophages were stimulated by LPS. AsBe alone
(without LPS) also did not induce the release of TNFα from
macrophages (data not shown).
Appl. Organometal. Chem. 2004; 18: 431–437
435
Bioorganometallic Chemistry
A
M-Mps
120
AsBe
arsenate
100
a
80
a
60
40
TNFα production
(pg per µg cellular protein)
T. Ohta T. Sakurai and K. Fujiwara
M-Mps
GM-Mps
25
20
15
10
5
0
0
b
20
100
1000
10000
AsBe concentrations (µmol dm-3)
0
0
100
B
1000
GM-Mps
120
Metabolic integrity (% Control)
436
10000
AsBe
arsenate
100
80
b
60
Figure 4.
Effect of AsBe on TNFα production from
monocyte-derived macrophages. Monocytes (2 × 104 per well)
were incubated with M-CSF ( ) or GM-CSF () on 96-well
tissue culture plates for 6 days at 37 ◦ C. After the incubation,
the cells were further exposed to various concentrations of AsBe
in the presence of 1 ng ml−1 LPS for 2 days at 37 ◦ C. TNFα
concentrations in the culture supernatants of monocyte-derived
macrophages ( , M-Mps; , GM-Mps) were measured by
ELISA. One representative experiment out of three similar
ones is shown. The results are expressed as arithmetic mean
plus/minus SEM of triplicate dishes.
°
°
b
40
b
20
60
Furthermore, we investigated the effects of AsBe on the
phagocytic ability of monocyte-derived macrophages and
on the T-cell stimulating ability of monocyte-derived DCs.
Macrophages or DCs differentiated from monocytes were
incubated with or without 5 mmol l−1 AsBe for 2 days at
37 ◦ C; the phagocytic activity of the macrophages and MLRs
of the DCs were then assessed. The results show that AsBe had
no effect on either phagocytic activity or T-cell stimulating
activity of these mature immune effector cells (data not
shown).
40
Cellular arsenic uptake in human monocytes
0
0
100
C
1000
DCs
120
10000
AsBe
arsenate
100
a
80
20
b
0
0
100
1000
10000
Arsenic concentrations (µmol dm-3)
Figure 3. Cytolethality of arsenic on the monocyte-derived
macrophages and DCs. Monocytes (2 × 104 per well) were
incubated with M-CSF (A), GM-CSF (B) or GM-CSF plus IL-4
(C) on 96-well tissue culture plates for 6 days at 37 ◦ C. After
the incubation, the cells were further exposed to various
concentrations of AsBe ( ), arsenate () or medium alone
for more 2 days at 37 ◦ C. The cellular viability of the cells (A,
M-Mps; B, GM-Mps; C, DCs) was determined by AB assay.
One representative experiment out of five similar ones is shown.
The results are expressed as arithmetic mean plus/minus SEM
of triplicate dishes. a p < 0.01 comparison with control cells
incubated with medium alone. b p < 0.001.
°
Copyright  2004 John Wiley & Sons, Ltd.
Monocytes (1 × 106 per well) were incubated with 5 mmol l−1
AsBe or arsenate for 24 h, and the arsenic amount in the
cell lysates was determined by hydride generation coupled
with atomic absorption spectrometry. As a result, the cellular
uptake of AsBe in human monocytes was about 10-fold lower
than that of arsenate; when monocytes were exposed to
arsenate, 4106 ± 77.0 µg of arsenic per gram cellular protein
was detected from the cell lysates, but only 456.3 ± 97.8 µg
of arsenic per gram cellular protein was detected when
monocytes were incubated with AsBe.
DISCUSSION
Marine animals, containing very high concentrations of
arsenicals,2 – 4 are ingested daily as seafood in many countries
in the world. These arsenicals are commonly in the form
of water-soluble organic arsenic compounds metabolized
Appl. Organometal. Chem. 2004; 18: 431–437
Bioorganometallic Chemistry
by marine animals from inorganic arsenic. In the marine
ecosystem, it has been demonstrated that inorganic arsenic in
sea water is probably taken up into seaweed and metabolically
methylated to dimethylarsinoyl ribosides (arsenosugars).17
Arsenosugars are further converted into trimethyl arsenic
compounds in many species of marine animal. Most of the
trimethyl arsenic contained in marine animals is AsBe,4 and
AsBe is thought to be the final metabolite in the arsenic
cycle of marine ecosystems because it is widely distributed
in various species of marine animal. Many people take in
considerable amounts of AsBe through ingesting seafood.
Thus, it is very important for us to investigate the biological
effects of AsBe on the human living system. However, there
have been only a few reports about those biological effects. In
this study, we examined whether AsBe has any toxicological
and/or biological effects on human immune effector cells,
such as human peripheral blood monocytes, macrophages
and DCs, which are known to be very sensitive to changes in
environmental conditions.12,15,16,18
One of the principal characteristics of monocytes is
to differentiate into some mature immune effector cells,
such as macrophages and DCs.9,10 In this study, we
investigated the effects of AsBe on the differentiation of
monocytes into macrophages or DCs in vitro and compared
this with the effects of inorganic arsenate. As shown in
Figs 1 and 2, AsBe had no effect on the number and
cellular viabilities of monocyte-derived macrophages or
DCs, although arsenate showed significant cytolethality in
them at micromolar concentrations. As shown in Tables 1
and 2, AsBe did not affect the principal functions of
monocyte-derived macrophages or DCs. Laupeze et al.19
reported that polycyclic aromatic hydrocarbons, known to
be environmental carcinogens, such as benzo(a)pyrene (BP),
had immunotoxicity on differentiation and maturation of
monocyte-derived DCs; 10 µmol l−1 BP exposure during
monocyte differentiation into DCs by GM-CSF plus IL-4
markedly decreased endocytic activity and T-cell stimulating
activity. These data imply that the experimental model using
differentiation of monocytes into DCs is capable of evaluating
the immunotoxicities of lower concentrations of chemicals
having no cytolethality. Thus, this assay, using differentiation
of monocytes into macrophages or DCs, is believed to be more
sensitive for evaluating chemical cytolethality than an assay
using simple cytolethality. It is suggested that AsBe had no
toxicological effects on the human living system.
The reasons why AsBe shows no cytolethality in human
immune cells has not yet been clarified. However, we
demonstrated in this study that the cellular uptake of a
pentavalent organic arsenic, AsBe, was markedly lower than
that of a toxic pentavalent inorganic arsenic, i.e. arsenate.
Thus, hereafter, it is necessary to investigate the relationship
between the cellular uptake and the chemical forms of arsenic
compounds.
In 2001 we found that AsBe significantly enhanced the
initial adhesion ability and viability of immature mouse BM
Copyright  2004 John Wiley & Sons, Ltd.
Arsenobetaine effects in human cells
cells and subsequently increased the continuous survival
of Mac-1 (CD11b) positive large spreading cells, especially
granulocytes and macrophages, which originated from the
BM cells.8 This implied that AsBe had a possible application
as a biological response modifier (BRM) to increase the
number of granulocytes and macrophages. We previously
reported that AsBe had no acute in vitro cytolethality in
mouse macrophages, mouse lymphocytes, rat liver cells and
human skin cells.6 – 8,20,21 The present study shows that AsBe
neither enhanced nor inhibited the differentiation of human
monocytes into macrophages or DCs and did not disrupt the
major functions of monocyte-derived macrophages or DCs
exposed during their differentiations. Taken together, it may
be possible to use AsBe as a BRM without fatal toxic side
effects.
In summary, we have demonstrated that AsBe was
not very cytotoxic in human monocytes, monocyte-derived
macrophages and DCs. AsBe is the majority component of the
arsenic contained in marine animals that are ingested daily
as seafood in many countries. Thus, the present study has
significance in food hygiene.
Acknowledgements
We express our thanks to Dr Masumi H. Sakurai (Azabu University,
Japan) for the limulus test and help with the preparation of this
manuscript, and to Mr Norihumi Tomita and Miss Yukie Hariya for
their excellent technical assistance.
REFERENCES
1. Morton WE, Dunnette DA. In Arsenic in the Environment Part II:
Human Health and Ecosystem Effects, Nriagu JO (ed.). John Wiley
and Sons: New York, 1994; 17–34.
2. Kaise T, Hanaoka K, Tagawa S, Hirayama T, Fukui S. Appl.
Organometal. Chem. 1988; 2: 539.
3. Le X-C, Cullen WR, Reimer KJ. Clin. Chem. 1994; 40: 617.
4. Edmonds JS, Francesconi KA, Cannon JR, Raston CR, Skelton BW, White AH. Tetrahedron Lett. 1977; 18: 1543.
5. Kaise T, Watanabe S, Itoh K. Chemosphere 1985; 14: 1327.
6. Sakurai T, Kaise T, Matsubara C. Appl. Organometal. Chem. 1996;
10: 727.
7. Kojima C, Sakurai T, Ochiai M, Kumata H, Qu W, Maalkes MP,
Fujiwara K. Appl. Organometal. Chem. 2002; 16: 421.
8. Sakurai T, Fujiwara K. Br. J. Pharmacol. 2001; 132: 143.
9. Akagawa KS, Takasuka N, Nozaki Y, Komuro I, Azuma M,
Ueda M, Naito M, Takahashi K. Blood 1996; 88: 4029.
10. Akagawa KS. Int. J. Hematol. 2002; 76: 27.
11. Banchereau J, Steinman RM. Nature 1998; 392: 245.
12. Sakurai T, Kaise T, Matsubara C. Chem. Res. Toxicol. 1998; 11: 273.
13. Sakurai T, Qu W, Sakurai MH, Waalkes MP. Chem. Res. Toxicol.
2002; 15: 629.
14. Thomas R, Davis LS, Lipsky PE. J. Immunol. 1993; 151: 6840.
15. Mosser DM. J. Leukoc. Biol. 2003; 73: 209.
16. Issekutz AC, Issekutz TB. J. Immunol. 1993; 151: 2105.
17. Edmonds JS, Francesconi KA. Nature 1981; 289: 602.
18. Manome H, Aiba S, Tagami H. Immunology 1999; 98: 481.
19. Laupeze B, Amiot L, Sparfel L, Ferrec EL, Fauchet R, Fardel O.
J. Immunol. 2002; 168: 2652.
20. Sakurai T. Appl. Organometal. Chem. 2002; 16: 401.
21. Sakurai T, Kojima C, Ochiai M, Ohta T, Fujiwara K. Int.
Immunopharmacol. 2004; 4: 179.
Appl. Organometal. Chem. 2004; 18: 431–437
437